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Culture of human cell lines by a pathogen-inactivated human platelet lysate

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Abstract

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

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References

  • Avanzini MA, Bernardo ME, Cometa AM, Perotti C, Zaffaroni N, Novara F, Visai L, Moretta A, Del Fante C, Villa R, Ball LM, Fibbe WE, Maccario R, Locatelli F (2009) Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors. Haematologica 94:1649–1660

  • Baik SY, Lim YA, Kang SJ, Ahn SH, Lee WG, Kim CH (2014) Effects of platelet lysate preparations on the proliferation of HaCaT cells. Ann Lab Med 34:43–50

    Article  Google Scholar 

  • Balk SD, Levine SP, Young LL, LaFleur MM, Raymond NM (1981) Mitogenic factors present in serum but not in plasma. Proc Natl Acad Sci USA 78:5656–5660

    Article  CAS  Google Scholar 

  • Balls M, Morton D (2010) FRAME and the three Rs: yesterday, today and tomorrow. Altern Lab Anim 38:269–274

    CAS  Google Scholar 

  • Barsotti MC, Losi P, Briganti E, Sanguinetti E, Magera A, Al Kayal T, Feriani R, Di Stefano R, Soldani G (2013) Effect of platelet lysate on human cells involved in different phases of wound healing. PLoS One 8:e84753

  • Bernardi M, Albiero E, Alghisi A, Chieregato K, Lievore C, Madeo D, Rodeghiero F, Astori G (2013) Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells. Cytotherapy 15:920–929

  • Bernardo ME, Avanzini MA, Perotti C, Cometa AM, Moretta A, Lenta E, Del Fante C, Novara F, de Silvestri A, Amendola G, Zuffardi O, Maccario R, Locatelli F (2007) Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. J Cell Physiol 211:121–130

  • Bieback K, Hecker A, Kocaomer A, Lannert H, Schallmoser K, Strunk D, Kluter H (2009) Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27:2331–2341

    Article  CAS  Google Scholar 

  • Brunner D, Frank J, Appl H, Schoffl H, Pfaller W, Gstraunthaler G (2010) Serum-free cell culture: the serum-free media interactive online database. Altex 27:53–62

    Google Scholar 

  • Castiglia S, Mareschi K, Labanca L, Lucania G, Leone M, Sanavio F, Castello L, Rustichelli D, Signorino E, Gnetti M, Bergallo M, Bordiga AM, Ferrero I, Fagioli F (2014) Inactivated human platelet lysate with psoralen: a new perspective for mesenchymal stromal cell production in Good Manufacturing Practice conditions. Cytotherapy 16:750–763

  • Cholewa D, Stiehl T, Schellenberg A, Bokermann G, Joussen S, Koch C, Walenda T, Pallua N, Marciniak-Czochra A, Suschek CV, Wagner W (2011) Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density. Cell Transpl 20:1409–1422

  • Doucet C, Ernou I, Zhang Y, Llense JR, Begot L, Holy X, Lataillade JJ (2005) Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol 205:228–236

    Article  CAS  Google Scholar 

  • Fekete N, Rojewski MT, Furst D, Kreja L, Ignatius A, Dausend J, Schrezenmeier H (2012) GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC. PLoS One 7:e43255

    Article  CAS  Google Scholar 

  • Ferro F, Spelat R, Beltrami AP, Cesselli D, Curcio F (2012) Isolation and characterization of human dental pulp derived stem cells by using media containing low human serum percentage as clinical grade substitutes for bovine serum. PLoS One 7:e48945

    Article  CAS  Google Scholar 

  • Gstraunthaler G (2003) Alternatives to the use of fetal bovine serum: serum-free cell culture. Altex 20:275–281

    Google Scholar 

  • Hankey DP, McCabe RE, Doherty MJ, Nolan PC, McAlinden MG, Nelson J, Wilson DJ (2001) Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum. Acta Orthop Scand 72:395–403

    Article  CAS  Google Scholar 

  • Hemeda H, Giebel B, Wagner W (2014) Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells. Cytotherapy 16:170–180

    Article  CAS  Google Scholar 

  • Hofbauer P, Riedl S, Witzeneder K, Hildner F, Wolbank S, Groeger M, Gabriel C, Redl H, Holnthoner W (2014) Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells. Cytotherapy 16:1238–1244

  • Honn KV, Singley JA, Chavin W (1975) Fetal bovine serum: a multivariate standard. Proc Soc Exp Biol Med 149:344–347

    Article  CAS  Google Scholar 

  • Ishiyama K, Satoh S, Igarashi Y, Kumagai H, Yahagi A, Sasaki H (1994) Flow cytometric analysis of the cell cycle of the leukemic cell lines treated with etoposide and cytosine arabinoside. Tohoku J Exp Med 174:95–107

    Article  CAS  Google Scholar 

  • Iudicone P, Fioravanti D, BonannoG, Miceli M, Lavorino C, Totta P, Frati L, Nuti M, Pierelli L (2014) Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells. J Transl Med 12:28

  • Jochems CE, van der Valk JB, Stafleu FR, Baumans V (2002) The use of fetal bovine serum: ethical or scientific problem? Altern Lab Anim 30:219–227

    CAS  Google Scholar 

  • Johansson L, Klinth J, Holmqvist O, Ohlson S (2003) Platelet lysate: a replacement for fetal bovine serum in animal cell culture? Cytotechnology 42:67–74

    Article  CAS  Google Scholar 

  • Kocaoemer A, Kern S, Kluter H, Bieback K (2007) Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells 25:1270–1278

    Article  CAS  Google Scholar 

  • Lin HT, Tarng YW, Chen YC, Kao CL, Hsu CJ, Shyr YM, Ku HH, Chiou SH (2005) Using human plasma supplemented medium to cultivate human bone marrow-derived mesenchymal stem cell and evaluation of its multiple-lineage potential. Transpl Proc 37:4504–4505

  • Morimoto N, Takemoto S, Kanda N, Ayvazyan A, Taira MT, Suzuki S (2011) The utilization of animal product-free media and autologous serum in an autologous dermal substitute culture. J Surg Res 171:339–346

    Article  CAS  Google Scholar 

  • Rauch C, Feifel E, Amann EM, Spotl HP, Schennach H, Pfaller W, Gstraunthaler G (2011) Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media. Altex 28:305–316

    Article  Google Scholar 

  • Reed GL (2004) Platelet secretory mechanisms. Semin Thromb Hemost 30:441–450

    Article  CAS  Google Scholar 

  • Schallmoser K, Bartmann C, Rohde E, Reinisch A, Kashofer K, Stademeyer E, Drexler C, Lanzer G, Linkesch W, Strunk D (2007) Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47:1436–1446

  • Schallmoser K, Strunk D (2013) Generation of a pool of human platelet lysate and efficient use in cell culture. Methods Mol Biol 946:349–362

    Article  CAS  Google Scholar 

  • Stute N, Holtz K, Bubenheim M, Lange C, Blake F, Zander AR (2004) Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use. Exp Hematol 32:1212–1225

    Article  CAS  Google Scholar 

  • Sundin M, Ringden O, Sundberg B, Nava S, Gotherstrom C, Le Blanc K (2007) No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica 92:1208–1215

    Article  CAS  Google Scholar 

  • Trojahn Kolle SF, Olivieri RS, Glovinski PV, Kirchhoff M, Mathiasen AB, Elberg JJ, Andersen PS, Drzewiecki KT, Fischer-Nielsen A ( 2013) Pooled human platelet lysate versus fetal bovine serum-investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use. Cytotherapy 15:1086–1097

  • Warnke PH, Humpe A, Strunk D, Stephens S, Warnke F, Wiltfang J, Schallmoser K, Alamein M, Bourke R, Heiner P, Liu Q (2013) A clinically-feasible protocol for using human platelet lysate and mesenchymal stem cells in regenerative therapies. J Craniomaxillofac Surg 41:153–161

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Acknowledgments

This work was supported in part by a grant of Futura Stem Cells sa.

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Authors and Affiliations

  1. InScientiaFides Foundation, San Marino, Republic of San Marino

    R. Fazzina

  2. Immunohematology and Transfusion Medicine, San Camillo-Forlanini Hospital, Rome, Italy

    P. Iudicone, D. Fioravanti, E. Cicchetti & L. Pierelli

  3. Institute of Gynecology and Obstetrics, Sacred Heart Catholic University, Rome, Italy

    A. Mariotti, A. Procoli, G. Scambia & G. Bonanno

  4. Department of Experimental Medicine, Sapienza University, Rome, Italy

    L. Pierelli

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  1. R. Fazzina
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  2. P. Iudicone
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  3. A. Mariotti
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  4. D. Fioravanti
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  5. A. Procoli
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  6. E. Cicchetti
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  7. G. Scambia
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  8. G. Bonanno
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  9. L. Pierelli
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Correspondence to L. Pierelli.

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Fazzina, R., Iudicone, P., Mariotti, A. et al. Culture of human cell lines by a pathogen-inactivated human platelet lysate. Cytotechnology 68, 1185–1195 (2016). https://doi.org/10.1007/s10616-015-9878-5

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  • DOI: https://doi.org/10.1007/s10616-015-9878-5

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